Use of insulin-like growth factor binding protein 3 (IGF-BP3) for inhibition of tumor growth

ABSTRACT

A method of inhibiting proliferation of cells associated with a tumor in a subject which comprises administering to the subject a tumor cell proliferation amount of IGF-BP3, thereby inhibiting proliferation of the cells. An improved surgical method which comprises surgically resecting a tumor from a subject and administering to the subject an amount of a protein effective to inhibit metastasis of any tumor cells released in the subject&#39;s blood circulation during the surgical resection of the tumor.

This application claims benefit of U.S. Provisional Application No.60/357,000, filed Feb. 13, 2002, the contents of which are herebyincorporated by reference.

Throughout this application, various publications are referenced by thefirst author's last name in parenthesis. Full citations for thesepublications may be found at the end of the specification immediatelypreceding the claims. The disclosures of these publications in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art as known to thoseskilled therein as of the date of the invention described and claimedherein.

BACKGROUND OF THE INVENTION

The mainstay of treatment for the vast majority of intestinal andvisceral malignancies has been “radical” resection of the tumor vialaparotomy. In the past decade an alternative abdominal access method,namely laparoscopy, has been utilized, by some, for the curativeresection of malignancies. This use of minimally invasive methodsremains controversial because of the lack of long-term studies andconcerns about port wound tumors. Early results from randomized trialscomparing traditional to laparoscopic-assisted colon resection forcancer have shown that an adequate minimally invasive oncologicresection can be done (Milsom, J. W., et al.).

Numerous experimental studies have demonstrated that laparotomy, whencompared to C0₂ pneumoperitoneum or anesthesia alone, is associated withincreased rates of tumor establishment and growth (Shiromizu, A., etal.; Allendorf, J. D., et al., 1995; Southall, J. C., et al.; Lee, S.W., et al., 1999). Similar results were noted after open and closedbowel resection (Allendorf, J. D., et al., 1998). Tumor cellproliferation was shown to be increased and apoptosis decreased afterlaparotomy in a murine study (Lee, S. W., et al., 1998). The mechanismof these tumor growth differences has also been investigated. Laparotomyrelated inhibition of immune function may account for some of theobserved differences in tumor growth after surgery (Allendorf, J. D., etal., 1999; Da Costa, M. L., et al.). Laparotomy associated elevation ofcirculating active protein substances, such as VEGF may also play a role(Pidgeon, G. P., et al.). In an animal study that assessed the abilityof pre- and postoperative mouse plasma to support tumor cells in vitro,significantly greater growth was noted in cultures to which postlaparotomy serum (from postoperative days 2 and 4) had been added (Lee,S. W., et al., 2000). It was postulated that a surgery-related plasmafactor accounted for the differences observed. In another study donewith the same model, the factor was identified as platelet derivedgrowth factor (PDGF) (Lee, S. W., et al., 2001)

SUMMARY OF THE INVENTION

The subject invention provides a method of inhibiting the proliferationof cells associated with a tumor in a subject which comprisesadministering to the subject a tumor cell proliferation amount ofIGF-BP3, thereby inhibiting proliferation of the cells.

The subject invention further provides an improved surgical method whichcomprises surgically resecting a tumor from a subject and administeringto the subject an amount of a protein effective to inhibit metastasis ofany tumor cells released in the subject's blood circulation during thesurgical resection of the tumor.

The improved surgical method comprises a surgical procedure on a subjectand administering to the subject a prophylactically effective amount ofIGF-BP3 to prevent proliferation of a tumor cell in the subject.

The subject invention also provides an article of manufacture comprisingpackaging material, IGF-BP3, and instructions for use of the IGF-BP3 ina surgical procedure.

DESCRIPTION OF THE FIGURES

FIG. 1. Correlation between the Increase in OS Plasma Mitogenic Activityon POD1 and the Length of Incision. HT29 cells were incubated with 10%plasma from patients undergoing open surgery and BrdU incorporation testperformed. A percentage increase in BrdU+ cells on POD1 versus preOP wascalculated and plotted to the length of the incision.

FIG. 2. IGF-BP3 Western Blots. Each pair of lanes is one patient's PreOPand POD1 results; Lanes 1 & 2 show an OS patient; Lanes 3 & 4 and 5 & 6,respectively, are 2 LS patients' results. IGF-BP3 was notably decreasedin the OS patient on POD1 (Lane 2) versus PreOP (Lane 1), but not in theLS patients (Lanes 4 and 6 vs. Lanes 3 and 5).

FIG. 3. Direct Inhibitory Effect of IGF-BP3 on Growth of Colon CancerCells. HT29 cells were plated in serum free conditions with IGF-BP3 invarious concentrations. The resulting number of recovered cells (left)and the percentage of BrdU+ cells in cultures decreased with increasingconcentrations of IGF-BP3.

FIG. 4. Neutralization of the Mitogenic Effect. Each triplet displaysone patient's results (unshaded bars, PreOP results; black bars, POD1results; crosshatched bars, rhIGFBP3 supplemented POD1 results).Recombinant human IGF-BP3 was added to cell cultures containing 10% POD1OS plasma. The percentage of BrdU+ cells and the total cell count weredecreased in supplemented wells (p<0.05 vs. PreOp Plasma) compared toresults with POD1 plasma alone (closed). RhIGF-BP3 POD1 OS plasma vs.PreOP OS plasma, no difference noted.

FIG. 5. Impact of Anti-IGFBP3 Antibody on the Mitogenic Effect of PreOPOS Plasma. Each triplet displays one patient's results (unshaded bars,PreOP results; crosshatched bars, ab+PreOp Plasma results; black bars,POD1 OS results). Neutralizing antibody to IGF-BP3 was added to wellscontaining PreOP OS plasma (concentration per well 10 μg/ml). HT29proliferation (counts and BrdU incorporation) was significantly higherin antibody supplemented wells when compared to PreOP OS plasma results(p<0.05). The addition of antibody raised PreOp Plasma associated HT29proliferation to levels observed with the POD1 OS plasma.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention provides a method of inhibiting proliferation ofcells associated with a tumor in a subject which comprises administeringto the subject a tumor cell proliferation amount of IGF-BP3, therebyinhibiting proliferation of the cells.

This invention further provides an improved surgical method whichcomprises surgically resecting a tumor from a subject and administeringto the subject an amount of a protein effective to inhibit metastasis ofany tumor cells released in the subject's blood circulation during thesurgical resection of the tumor.

The improved surgical method which comprises a surgical procedure on asubject and administering to the subject a prophylactically effectiveamount of IGF-BP3 to prevent proliferation of a tumor cell in thesubject.

In an embodiment of the invention, the tumor is associated with coloncancer, prostate cancer, breast cancer or lung cancer.

In specific embodiments, the subject is human and IGF-BF3 is recombinantIGF-BP3.

In another embodiment of the invention, the administration of IGF-BP3 isoral, intravenous or transdermal.

In an embodiment, the administration of IGF-BP3 is intravenous.

In a further embodiment of the invention, the surgical procedure is openabdominal surgery.

In an embodiment of the immediately preceding, the administration ofIGF-BP3 is before start of the surgery.

In yet another embodiment of the preceding, the administration ofIGF-BP3 is concurrent with the surgery.

In yet another embodiment of the preceding, the administration ofIGF-BP3 is after the completion of the surgery.

In a specific embodiment of the preceding, the surgical procedure iscolorectomy or gastric bypass.

The subject invention also provides an article of manufacture comprisingpackaging material, IGF-BP3, and instructions for use of the IGF-BP3 ina surgical procedure.

In an embodiment, the process of manufacturing the article comprisescombining the packaging material, the IGF-BP3, and the instructions soas to manufacture the article.

The invention also provides the use of IGF-BP3 in the manufacture of apharmaceutical composition for administering to a subject to inhibitproliferation of cells associated with a tumor.

In an embodiment of the preceding, IGF-BP3 is used in the manufacture ofa pharmaceutical composition for administering to a subject before,during, or after a surgical procedure resecting a tumor from the subjectin an amount effective to inhibit metastasis of any tumor cells releasedin the subject's blood circulation during the surgical resection of thetumor.

In a further embodiment of the preceding, IGF-BP3 is used in themanufacture of a pharmaceutical composition for administering to asubject before, during, or after a surgical procedure in an amounteffective to prevent proliferation of a tumor cell in the subject.

This invention also provides an improved surgical use which comprises asurgical procedure on a subject and administering to the subject aprophylactically effective amount of IGF-BP3 to prevent proliferation ofa tumor cell in the subject.

In an embodiment of the immediately preceding, IGF-BP3 is used fortumors associated with colon cancer, prostate cancer, breast cancer orlung cancer.

In a specific embodiment, recombinant IGF-BP3 is used for a humansubject.

In an embodiment, the use of IGF-BP3 is via oral, intravenous ortransdermal administration.

In an embodiment, the use of IGF-BP3 is via intravenous administration.

In one embodiment, the use of IGF-BP3 is in a surgical procedure whereinthe surgical procedure is open abdominal surgery.

In a further embodiment, the use of IGF-BP3 is before start of thesurgery.

In a yet a further embodiment, the use of IGF-BP3 is concurrent with thesurgery.

In yet a further embodiment, the use of IGF-BP3 is after the completionof the surgery.

In a specific embodiment, the use of IGF-BP3 is for surgical procedurewherein the surgical procedure is colorectomy or gastric bypass.

The term “recombinant human antibody,” as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library,antibodies isolated from an animal that is transgenic for humanimmunoglobulin genes or antibodies (Taylor, L. D., et al.) or antibodiesprepared, expressed, created or isolated by any other means thatinvolves splicing of human immunoglobulin gene sequences to other DNAsequences. Such recombinant human antibodies have variable and constantregions derived from human germline immunoglobulin sequences.

The antibodies and antibody-portions of the invention can beincorporated into pharmaceutical compositions suitable foradministration to a subject. Typically, the pharmaceutical compositioncomprises an antibody (or antibody portion) of the invention and/ormethotrexate and a pharmaceutically acceptable carrier.“Pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible and are suitable for administration to asubject. Examples of pharmaceutically acceptable carriers include one ormore of water, saline, phosphate buffered saline, dextrose, glycerol,ethanol and the like, as well as combinations thereof. In many cases, itwill be preferable to include isotonic agents, for example, sugars,polyalcohols such as mannitol, sorbitol, or sodium chloride in thecomposition. Pharmaceutically acceptable carriers may further compriseminor amounts of auxiliary substances such as wetting or emulsifyingagents, preservatives or buffers, which enhance the shelf life oreffectiveness of the antibody or antibody portion.

The compositions of the invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, the antibody is administered by intravenous infusion orinjection. In another preferred embodiment, the antibody is administeredby intramuscular injection. In a particularly preferred embodiment, theantibody is administered by subcutaneous injection.

The antibodies and antibody-portions of the present invention can beadministered by a variety of methods known in the art, although for manytherapeutic applications, the preferred route/mode of administration issubcutaneous injection. As will be appreciated by the skilled artisan,the route and/or mode or administration will vary depending upon thedesired results. In certain embodiments, the active compound may beprepared with a carrier that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyethylene glycol (PEG), polyanhydrides, polyglycolicacid, collagen, polyorthoesters, and polylactic acid. Many methods forthe preparation of such formulations are patented or generally known tothose skilled in the art. See, e.g., Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978.

In certain embodiments, an antibody or antibody portion of the inventionmay be orally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafersand the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Experimental Details

Materials

Patients. Eighty-four patients (43 males and 42 females) that underwenteither a colorectal resection or a gastric bypass for morbid obesitywere included in this study. Forty-five patients underwent open surgerywhereas 39 had minimally invasive procedures. Patients oncorticosteroids, other immunosuppressive drugs, and those who hadundergone chemotherapy or radiotherapy within 3 months of surgery wereexcluded from the study. Anesthesia was induced and maintained withpropofol, succinyl choline and nitrous oxide. Additionally fentanyl andmagnesium sulfate were given. The study was approved by theinstitutional IRB and informed consent was obtained from all patients.TABLE 1 Indications for Surgery Indication OS group, n (%) LS group, n(%) Colorectal cancer 20 (44) 22 (56)  Morbid Obesity 13 (29) 8 (21)Colorectal Adenoma  8 (18) 5 (13) Diverticular Disease 4 (9) 4 (10)OS denotes Open SurgeryLS denotes Laparoscopic Surgery

TABLE 2 Accompanying Diseases in LS and OS Patients LS group, nAccompanying Diseases OS group, n (%) (%) Coronary artery disease,Hypertension 14 (31) 16 (41) Chronic Obstructive Pulmonary  5 (11) 3 (8)Disease, Asthma Diabetes 4 (9) 2 (5) Past surgery  5 (11)  4 (10)

The indications for surgery are provided in Table 1 whereas Table 2concerns associated illnesses. The two groups were statistically similarin regards to indication and associated illnesses, age (overall groupsand subgroups), and mean height and weight for each group. Furthermore,in regards to the colon cancer patients, there were no significantdifferences noted in final tumor stage, size of tumor, overall length ofspecimen, number of lymph nodes, or margins. There were no conversionsin the laparoscopic group. None of the patients in either group receivedperioperative blood transfusions. The mean length of incision was19.4±4.7 cm in the OS group and 5.0±2.1 cm in LS group.

Peripheral Blood Collection. Samples were collected in EDTA containingtubes pre-operatively and on post-operative days 1 and 3. Plasma wasisolated by centrifugation soon after being drawn and stored at −80° C.until used.

Tumor Cell Line. The HT29 tumor, a human colonic adenocarcinoma cellline, was obtained from ATCC (Manassas, Va.) and maintained in completeDMEM medium (Cellgro, Herndon, Va.) with 10% fetal calf serum(FCS)(Cellgro). For the assay, HT29 cells were plated in 6-well plates,2×10⁵ cells/well in 2 ml of complete medium with FCS, and allowed toadhere. Cells were then washed 2 times with serum free DMEM medium andincubated for 48 hours with 10% human serum from the patients.

Biological Testing

5-Bromo-deoxyuridine (BrdU) Incorporation Assay. Two hours before theend of the incubation period, cells were pulsed with BrdU (BDPharmingen, San Diego, Calif.), in a final concentration of 10 μM. Cellswere then harvested by trypsinization, counted and washed with PBS 3times. Cells were then fixed in 70% ethanol, washed again and denaturedwith 2M HCl. After neutralization with 0.1M sodium borate solution and 3washes, cells were incubated with FITC labeled monoclonal antibody toBrdU (Caltag, Burlingame, Calif.), washed again and analyzed by flowcytometry using FACS Calibur (Becton Dickinson).

Total Cell Count. The total number of viable tumor cells in finalcultures was determined by trypan blue dye (Cellgro) exclusion.

Detection of IGF-BP3 by Western Blot Analysis. Plasma, 5 μl diluted inTris-Glycine loading buffer was electrophoresed on 18% Tris-Glycinepre-cast gels (Invitrogen, Carlsbad, Calif.) and transferred to asupported nitrocellulose membrane (Bio-Rad, Hercules, Calif.). Membraneswere then blocked with 3% milk, incubated with a polyclonal biotinylatedantibody to human IGF-BP3 (R&D Systems, Minneapolis, Minn.), washed withPBS, incubated with peroxidase labeled streptavidin (BD Pharmingen) andwashed again. Membranes were developed using ECL reagent(Buckinghamshire, England) and an X-ray film.

Other IGF-BP3 Experiments. Neutralizing antibody to IGF-BP3 (humanIGFBP3 specific goat IgG produced in goats immunized with purified,NSO-derived, recombinant IGFBP3): (R&D Systems, Minneapolis, Minn.) wasadded to the tumor cell cultures in final concentration 10 μg/ml.Recombinant purified human IGF-BP3 (rhIGF-BP3: Purified withPhenyl-Sepharose chromatography, Gel Filtration, IGFBP3 affinitychromatography, Reverse Phase HPLC; MW=47,000 Da) (UpstateBiotechnology, Lake Placid, N.Y.) was added to HT29 cells plated aspreviously described in final concentration 100-750 ng/ml in serum freemedium. When added to wells containing 10% human serum, rhIGF-BP3 wasadded in final concentration 750 ng/ml.

Statistical Analysis. Data are expressed as means±SD. Wilcoxon'smatched-pairs signed-ranks test and Spearman's correlation tests wereused for statistical analysis.

Results

In Vitro Tumor Cell Proliferation TABLE 3 Mitogenic Activity of Plasmafrom the OS and LS Groups Plasma Mitogenic Activity Cells recovered AgeBrdU + cells, % from culture ×10⁵ Patient Groups N Yrs. PreOP POD1 PreOPPOD1 OS, All Patients 45 56.6 ± 15.8 34.2 ± 17.9 42.4 ± 19.5** 5.6 ± 1.67.0 ± 1.8** OS, Colon Cancer^(a) 20 65.4 ± 12.6 30.5 ± 19.1 36.3 ±18.0** 5.4 ± 1.7 6.7 ± 1.8** OS, Obesity 13 43.1 ± 10.9 48.5 ± 7.8 56.7± 6.4** 6.7 ± 0.6 7.8 ± 0.8** OS, Colon Adenoma 8 50.2 ± 15.0 23.7 ±14.3 33.8 ± 22.3* 4.3 ± 1.5 6.3 ± 3.0* OS, Diverticulitis 4 69.2 ± 6.2¶26.7 ± 17.4 43.9 ± 29.1¶ 5.7 ± 1.6 8.0 ± 1.7¶ LS, All Patients 39 59.8 ±19.3 37.2 ± 18.1 36.6 ± 18.9 5.2 ± 1.3 5.1 ± 1.7 LS, Colon Cancer^(a) 2263.9 ± 17.6 32.6 ± 19.9 31.6 ± 18.4 4.9 ± 1.4 4.8 ± 1.7 LS, Obesity 838.9 ± 14.7 47.0 ± 14.9 42.1 ± 15.9 5.9 ± 1.3 5.8 ± 1.6 LS, ColonAdenoma 5 74.8 ± 8.3▪ 49.6 ± 8.7 56.5 ± 16.5¶ 5.4 ± 1.0 5.8 ± 1.9¶ LS,Diverticulitis 4 60.8 ± 17.6¶ 34.9 ± 17.5 36.3 ± 23.3¶ 4.9 ± 0.7 5.0 ±0.8¶*P < 0.05;**P < 0.005 PreOP versus POD1 using Wilcoxon's matched-pairssigned-ranks test.▪p < 0.05 compared to identical OS subgroup.¶Insufficient n for a statistical analysis.^(a)Patients with colon cancer stage I-III were included; distributionof stages was comparable in OS and LS groups.

Open Surgery Group POD1 vs PreOp (Table 3). For the overall group, asignificantly higher proportion of tumor cells were BrdU+ in thecultures where POD1 OS plasma had been added (42.4±19.5%) when comparedto the PreOP OS plasma results (34.2±17.9%, p<0.005). In regards to thetotal number of viable tumor cells found at the end of the incubationperiod, significantly more cells (7.0±1.8×10⁵) were noted in the POD1 OSwells than in the PreOP OS wells (5.6±1.6×10⁵, p<0.005) when the OSgroup as a whole was considered. Similarly significant differences forboth proliferative parameters were noted when the colon cancer and themorbid obesity subgroups were considered separately. The other subgroupswere too small to permit statistical analysis. The increase in mitogenicactivity of POD1 OS plasma correlated with the length of surgicalincision (p<0.01, r=0.58) (FIG. 1). Thirteen OS POD3 plasma samples wereassessed. When considered together, no significant differences in BrdUincorporation or total cell count were noted when the POD3 and the PreOpplasma results were compared. However, when 5 patients with an incisionequal or greater than 23 cm were considered, significantly increasedHT29 proliferation was noted with the POD3 plasma when compared to thePreOP results (data not shown).

Laparoscopic Surgery Group. No differences in the percentage of BrdU+cells or the total number of tumor cells were noted when the POD1 LSdata were compared to the preOP LS data (Table 3). This was true for theoverall group and for the 2 main subgroups where analysis was possible.A total of 14 POD3 LS plasma samples were similarly assessed. Nodifferences in proliferation were noted when the POD3 LS and PreOp LSdata were compared.

Plasma Factor Characterization Studies

Initial Studies. In a subset of patients, blood samples were collectedin heparinized tubes in addition to the EDTA containing tubes. Althoughincreased HT29 proliferation was noted with POD1 OS vs PreOP OS EDTAsamples, no differences in tumor growth were noted when the heparinizedplasma was tested (data not shown). In addition, heating of plasma to99° C. eliminated the effect, which suggested that the factor(s) was aprotein. We searched for a protein serum factor which could bestabilized by EDTA. Insulin-like growth factor binding protein 3(IGF-BP3) was a likely candidate, because EDTA inhibits activity ofIGF-BP3 specific serum protease, an enzyme that cleaves IGF-BP3 (Bang,P., et al.). In addition, surgery has been reported to induce IGF-BP3protease activity (Davenport, M. L., et al.). Because the antibody inthe available ELISA for IGF-BP3 reacts both with the intact protein (−40kDa) and the biologically inert protein fragments, a Western Blotanalysis was performed.

Western Blot Analysis. PreOP and POD1 plasma levels of IGF-BP3 weredetermined for all patients. In 5 of 45 OS patients (11.1%) and 6 of 39LS patients (15.4%), IGF-BP3 was not detected in any of the samples. Forthe remaining patients, a decrease in plasma IGF-BP3 on POD1 whencompared to PreOP levels was noted in 80.9% of OS patients and in 16.7%of LS patients. OS patients with preserved post-operative levels ofcirculating IGF-BP3 had shorter incisions (<23 cm). RepresentativeWestern Blot results are displayed in FIG. 2.

IGF-BP3 Effect on HT 29 Growth. To test whether IGF-BP3 directly affectsHT29 growth, human recombinant IGF-BP3 (rhIGF-BP3) was added to serumfree cultures of HT29 cells (FIG. 3). RhIGF-BP3 had an inhibitory effecton HT29 cell proliferation in the concentration range of 200-750 ng/ml;higher concentrations have not been tested. IGF-BP3 concentrations lowerthan 200 ng/ml did not have an impact on cell proliferation.

RhIGF-BP3 was added to POD1 OS plasma samples (n=6) at concentration of750 ng/ml and HT29 proliferation assessed. Significantly lessproliferation, as judged by both BrdU assay and cell counts, was notedin the rhIGF-BP3 augmented wells (FIG. 4) when compared to POD1 OSplasma results. There was no significant difference between thesupplemented POD1 and the PreOP OS plasma results.

Impact of Neutralizing Antibody to IGF-BP3. Anti-IGF-BP3 antibody wasadded to PreOP OS plasma and HT29 proliferation assessed (n=6). BothBrdU and cell count results demonstrated significantly increasedproliferation for the antibody supplemented group when compared to thePreOP plasma results (p<0.05). The anti-IGF-BP3 results were similar tothose noted with the POD1 OS plasma (FIG. 5).

Discussion

The present human study was undertaken to determine if major open andclosed abdominal surgery had a similar effect on human plasma mitogenicactivity for colon cancer cells. In a murine model, open surgery inducedan increase in serum mitogenic activity and platelet-derived growthfactor was thought to be the responsible protein (Lee, S. W. et al.,2001). The purpose of this human study was to determine if majorabdominal surgery carried out via open or laparoscopic means wasassociated with alterations in the composition of plasma such that invitro tumor growth would be enhanced. If such an effect was indeedobserved, it was our hope to identify the responsible factor(s).

The results suggest that post-op day 1 plasma from open surgery patientsenhances in vitro tumor growth and that the increased proliferationcorrelated directly with the length of the incision. Post-operativeplasma from laparoscopic surgery patients did not have this effect.Increased growth was noted after both open colorectomy and gastricbypass and, therefore, does not appear to be related to the organ beingoperated on or to the presence of a malignancy. Although not randomized,close scrutiny of the open and closed groups in regards to demographics,associated illnesses, body habitus, indication, and pathology results(for tumors) suggested the two groups were similar. Of note,proliferation studies of the POD3 plasma, carried out on a fraction ofthe study patients, suggested that the tumor stimulatory effect is lostin all but those open patients with incisions equal or greater than 23cm.

Plasma from laparotomized mice has been shown to stimulate in vitrotumor growth when compared to results with preoperative plasma. Thisstudy assessed the effect of plasma from patients that underwent majoropen (OS) or laparoscopic surgery (LS) on in vitro tumor cell growth.Eighty-four patients undergoing major abdominal surgery were studied.(45 OS, 39 LS). Peripheral blood was collected preoperatively (PreOP)and on days 1 (POD1) and 3 (POD3) after surgery. HT29 human colon cancercells were plated with samples of the plasma.

Proliferation was assessed via cell counts and the BrdU incorporationassay. IGF-BP3 (insulin-like growth factor binding protein 3) wasdetected in plasma via Western Blot analysis. Increased mitogenicactivity was noted in POD1 OS plasma when compared to PreOP OS plasmaresults (p<0.005). This increase correlated with the length of incision(r=0.58, P<0.01). No differences were noted when the PreOP LS and POD1LS results were compared or for any of the POD3 vs PreOP comparisons.Hence, major open surgery is associated with alterations in plasmacomposition that promote HT29 tumor cell proliferation in vitro. Asshown, this effect was due, at least in part, to surgery-relateddepletion of IGF-BP3 in peripheral blood.

Plasma from patients undergoing major open surgery stimulates in vitrotumor growth. Lower IGF-BP3 levels may, in part, account for thischange. Plasma from mice undergoing laparotomy has been shown tostimulate in vitro tumor growth. The goals of this study were todetermine the effect of plasma from patients that underwent major open(OS) or laparoscopic surgery (LS) on in vitro tumor growth and, ifsurgery-related differences were noted, to identify the responsiblefactor(s). Materials: A total of 58 patients undergoing major abdominalsurgery were studied (34 OS and 24 LS patients). Peripheral blood wascollected in heparinized and EDTA tubes before surgery (PreOP) and ondays 1 (POD1) and 3 (POD3) after surgery. Plasma was obtained bycentrifugation and stored at −70° C.

Proliferation Assay: HT29 human colon cancer cells were plated with 10%human serum from the patients. The BrdU cell proliferation assay wasused. IGF-BP3 (insulin-like growth factor binding protein 3) wasdetected in plasma by Western Blot Analysis using specific antibody.Statistical Analysis was performed using paired Student's test andPearson correlation coefficient. [P value of 0.05 or less was consideredstatistically significant.]

Increased mitogenic activity was noted with the POD1 OS plasma(41.4±20.4% BrdU+ cells) when compared to results with the PreOP OSplasma (32.5%±17.9%, p<0.01). This increase correlated with the lengthof incision (r=0.328, P=0.036). No difference in mitogenic activity wasnoted when the LS PreOP and the LS POD1 results were compared(32.6±20.7% vs 31.7±20.8%, respectively). [No differences were notedwhen POD3 and PreOp results were compared for either the OS and LSgroups.] OS associated stimulation of HT29 cell growth was stronger withEDTA than with heparinized plasma. We searched for a serum factor thatmight be stabilized by EDTA. EDTA blocks IGF-BP3-related plasmaproteolytic activity. Via Western Blot analysis, less IGF-BP3 was notedin the POD1 plasma samples associated with higher mitogenic activity.Purified IGF-BP3 at a concentration 500 ng/ml and higher appeared toinhibit HT29 proliferation, while addition of IGF-BP3 neutralizingantibody to PreOP plasma increased its mitogenic activity to the levelof POD1 plasma. Major open surgery appears to enhance the ability ofhuman plasma to promote HT29 tumor cell proliferation in vitro. Thiseffect may be due, in part, to depletion of IGF-BP3 in peripheral bloodfollowing open surgery.

Having demonstrated the effect we next sought to identify theresponsible factor(s). Serum PDGF β levels were determined via ELISA,however, no differences were noted (data not provided) in either group.For the reasons stated, IGF-BP3 was a reasonable candidate. The WesternBlot findings and the results of the studies that supplemented withrhIGF-BP3 or anti-IGF-BP3 strongly suggest that IGF-BP3 is theresponsible factor. An open surgery-related decrease in IGF-BP3 levels,most likely, accounts, in large part, for the in vitro tumor growthdifferences noted with POD1 Open surgery plasma. It has previously beenshown that major open abdominal surgery induces proteolysis ofcirculating IGF-BP3 (Cotterill, A. M., et al.).

Intact IGF-BP3 can influence tumor growth via 2 mechanisms. First, itcan bind circulating IGF-I, a well known growth factor, and thus limitIGF-I related stimulatory effects (Yu, H., et al.). Secondly, IGF-BP3itself can deter proliferation directly by inducing tumor cellapoptosis. This direct effect has been documented for prostate cancer(Rajah, R., et al.), breast cancer (Fanayan, S., et al.) andhepatocellular carcinoma cells (Murakami, K., et al.). Interestingly,IGF-BP3's effect on colon cancer cells is less clear; one studysuggested it was inhibitory (MacDonald, R. G., et al.) while anotherreported stimulatory effects (Kansra, S., et al.). In this study, theHT29 colon cell line was shown to be inhibited by IGF-BP3.

The possible implications of these results are far reaching. For thefirst time, in humans, the choice of surgical approach has beenassociated with host alterations that may increase the chances thattumor cells in the blood will survive and form a metastases. Thus,IGF-BP3 replacement in open cancer patients or supplementation in closedsurgery patients should lower the risk of tumor recurrence.

Insulin-like growth factor binding protein 3 (IGF-BP3) is a serumprotein that can exert an inhibitory effect on the growth of tumor cellsvia 2 major mechanisms: 1) it binds a major cell growth factor,insulin-like growth factor 1 (IGF-1) and 2) it directly inhibits tumorcell growth. The inhibitory effect of IGF-BP3 has been demonstrated forprostate, breast, and lung cancer. We have shown that high doses ofIGF-BP3 also directly inhibits colon cancer cell growth in vitro.

In the described study, patients that underwent major open surgery(cancer and non-cancer patients) were noted to have reduced levels ofIGF-BP3 shortly after surgery. We found that plasma from open surgerypatients collected on postoperative day 1 stimulated in vitro tumorgrowth of colon cancer cells when compared to results obtained withtheir preoperative plasma samples. Addition of IGF-BP3 neutralizingantibody to preoperative plasma resulted in accelerated tumor cellgrowth to the same degree as observed with postoperative plasma. Inaddition, supplementing the postoperative day 1 plasma with recombinantIGF-BP3 eliminated the tumor cell stimulation that had been noted withthe “raw” postoperative day 1 plasma. Thus, we propose inhibition ofrecurrent tumor and/or metastatic tumor formation via perioperativeadministration of insulin-like growth factor binding protein 3 in cancerpatients (all types of cancer) undergoing major surgery via atraditional open (a single lengthy incision) surgical approach.

The surgical resection of cancers is associated with the release oftumor cells into the circulation in a significant proportion ofpatients. These blood borne tumor cells may give rise to distantmetastases. The chances that a circulating tumor cell will successfullyform a metastases, regardless of the cancer type, in a patientundergoing an open surgery will be smaller if immediately after surgerythe patient receives one of several injections of IGF-BP3.

Prior to our study, it was not known that open surgery induces thepartial depletion of circulating plasma IGF-BP3 and that the depletedpostoperative plasma stimulates cancer cell growth. Also, our studydemonstrates that a similar effect can be achieved by adding IGF-BP3neutralizing antibody to the preoperative plasma. Our study also showsthat higher concentrations of recombinant IGF-BP3 improve the inhibitoryeffect for, e.g., colon cancer cells.

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1. A method of inhibiting proliferation of cells associated with a tumorin a subject which comprises administering to the subject a tumor cellproliferation amount of IGF-BP3, thereby inhibiting proliferation of thecells.
 2. (canceled)
 3. The method of claim 1, wherein the tumor isassociated with colon cancer, prostate cancer, breast cancer or lungcancer.
 4. An improved surgical method which comprises a surgicalprocedure on a subject and administering to the subject aprophylactically effective amount of IGF-BP3 to prevent proliferation ofa tumor cell in the subject.
 5. The method of claim 1, wherein thesubject is human.
 6. The method of claim 1, wherein the IGF-BP3 isrecombinant IGF-BP3.
 7. The method of claim 1, wherein theadministration is oral, intravenous or transdermal.
 8. The method ofclaim 7 wherein the administration is intravenous.
 9. The method ofclaim 4 wherein the surgical procedure is open abdominal surgery. 10.The method of claim 4 wherein the administration of IGF-BP3 is beforestart of the surgery.
 11. The method of claim 4 wherein theadministration of IGF-BP3 is concurrent with the surgery.
 12. The methodof claim 4 wherein the administration of IGF-BP3 is after the completionof the surgery.
 13. The method of claim 4 wherein the surgical procedureis colorectomy or gastric bypass.
 14. An article of manufacturecomprising packaging material, IGF-BP3, and instructions for use of theIGf-BP3 in a surgical procedure.
 15. A process of manufacturing thearticle of claim 14 comprising combining the packaging material, theIGF-BP3, and the instructions so as to manufacture the article. 16-29.(canceled)
 30. The method of claim 4, wherein the subject is human. 31.The method of claim 4, wherein the IGF-BP3 is recombinant IGF-BP3. 32.The method of claim 4, wherein the administration is oral, intravenousor transdermal.
 33. The method of claim 31, wherein the administrationis intravenous.